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inducing calcium mobilization and stimulating cell migra- tion was examined in human transitional-cell carcinoma. (J82) cells. Measurements of cytoplasmic ...
Naunyn-Schmiedeberg’s Arch Pharmacol (1997) 356 : 769–776

© Springer-Verlag 1997

O R I G I N A L A RT I C L E

Gerd Lümmen · Sebastian Virchow · Ulrich Rümenapp · Martina Schmidt · Thomas Wieland · Thomas Otto · Herbert Rübben · Karl H. Jakobs

Identification of G protein-coupled receptors potently stimulating migration of human transitional-cell carcinoma cells

Received: 25 June 1997 / 8 August 1997

Abstract The expression of G protein-coupled receptors inducing calcium mobilization and stimulating cell migration was examined in human transitional-cell carcinoma (J82) cells. Measurements of cytoplasmic Ca2+ concentration ([Ca2+]i) and phospholipase C activity indicated that these cells express several calcium-mobilizing receptors, including those for lysophosphatidic acid (LPA), thrombin, bradykinin, bombesin and histamine, of which only the LPA response was sensitive (~ 50%) to pertussis toxin (PTX). Migration of J82 cells was strongly stimulated by LPA and thrombin, by 5- to 20-fold, whereas bradykinin, bombesin and histamine were ineffective. Migration induced by either LPA or thrombin was inhibited by the actin cytoskeleton-disrupting agent, cytochalasin B, by the Rho protein-inactivating Clostridium difficile toxin B, by preventing [Ca2+]i transients with an intracellular calcium-chelating agent, and by the phorbol ester, phorbol 12-myristate 13-acetate, which also blocked the LPA- and thrombin-induced [Ca2+]i increases. On the other hand, ADP-ribosylation of Gi type G proteins by PTX abrogated the migratory response to LPA, without affecting the thrombin effect. Similarly, raising cAMP levels inhibited, by about 50%, the LPA- but not the thrombin-induced J82 cell migration. In conclusion, human transitional-cell carcinoma (J82) cells express various G protein-coupled, calcium-mobilizing receptors, out of which only those for LPA and thrombin stimulate cell migration, indicating that phospholipase C-derived second messengers per se are not sufficient for initiating this response. The complex signal transduction processes leading to LPA- and thrombin-stimulated motility of these human carcinoma cells apparently involve several common, essential factors, such

G. Lümmen (Y) · S. Virchow · U. Rümenapp · M. Schmidt · T. Wieland · K. H. Jakobs Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany G. Lümmen · S. Virchow · T. Otto · H. Rübben Urologische Klinik und Poliklinik, Universitätsklinikum Essen, D-45122 Essen, Germany

as [Ca2+]i changes and Rho protein-regulated reorganization of the cytoskeleton, as well as some distinct components, most notably distinct subtypes of heterotrimeric G proteins and apparently also distinct cAMP-sensitive targets. Key words LPA · Thrombin · Migration · Bladder cancer cells · J82 cells

Introduction Migration of cells is a fundamental process for the life of multi-cellular beings and is involved in a wide variety of physiological and pathological processes, such as embryogenesis, wound healing, angiogenesis and inflammatory responses. In metastasis, malignant tumor cells migrate from the initial tumor site into the circulatory system, then invade a new site and disrupt normal tissue architecture, which accounts as much or more for the lethality of cancer than does uncontrolled growth (Liotta et al. 1991; Van Roy and Vareel 1992). Initiation of cell migration is caused by both substrate-bound and soluble extracellular molecules, interacting with specific cell surface receptors (Stoker and Gherardi 1991; Stossel 1993). Studies over the past years have elucidated some of the components and mechanisms participating in the signal transduction pathways that trigger cell migration, apparently involving the complex integration of various motility-promoting and motility-inhibiting signals. In particular, the dynamic changes in actin polymerization that contribute to cell migration (Korn 1978) and the regulation of these processes by the Rho family of small molecular weight GTP-binding proteins (G proteins) have been in the focus of recent interest (Hall 1994; Takai et al. 1995; Huttenlocher et al. 1995; Lauffenburger and Horwitz 1996; Zigmond 1996). Particularly, studies in 3T3 fibroblasts indicated that these G proteins, which include Rho, Rac and Cdc42, can be activated by receptors coupled to heterotrimeric G proteins and by tyrosine kinase receptors, and exhibit specific functions in migrating cells, by control-

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ling formation of filopodia, lamellipodia, focal adhesions and stress fibers. Although classical messengers, particular Ca2+, are generally considered important for motile responses (Korn 1978; Stossel 1993; Huttenlocher et al. 1995), they are apparently neither required for activation of Rho G proteins by membrane receptors nor for Rho family G protein-mediated rearrangements of the actin cytoskeleton (Hall 1994; Takai et al. 1995; Zigmond 1996). Inactivation of Rho G proteins results in collaps of the actin cytoskeleton, and the cells adopt a rounded morphology. Bladder cancers, one of the most common cancers and apparently increasing in incidence, are categorized as either invasive or superficial. Although over 70% of bladder cancer cases occur in superficial form, 20–30% do progress to invasive disease with a worse prognosis and a propensity to metastasize (Raghavan et al. 1990). One prognostic factor for bladder carcinomas is the expression of the epithelium-specific cell-cell adhesion molecule, Ecadherin, an invasion suppressor molecule, the expression of which is negatively correlated with cancer progression (Otto et al. 1994). Aim of the present study was to examine the expression of migration stimulatory receptors on bladder cancer cells. Specifically, the regulation of migration of J82 cells, a cell line originated from a human invasive transitional-cell carcinoma (O’Toole et al. 1978), by agonists acting on G protein-coupled receptors was studied. Therefore, we first examined the expression of G protein-coupled, calcium-mobilizing receptors in J82 cells and then studied whether activation of these endogenously expressed receptors induces cell migration as well as some basic mechanisms of this complex cellular response. We report there that these human carcinoma cells express calcium-mobilizing receptors for lysophosphatidic acid (LPA), thrombin, bradykinin, bombesin and histamine, and that of these ligands only LPA and thrombin potently stimulate cell motility, apparently by at least partially distinct mechanisms.

Materials and methods Materials. Minimal essential medium (MEM) with Earle’s salts, fetal calf serum, streptomycin, penicillin G and trypsin/EDTA were purchased from Gibco-BRL (Eggenstein, Germany). 1-OleoylLPA, bradykinin, bombesin, thrombin, histamine, phorbol 12-myristate 13-acetate (PMA), cytochalasin B, forskolin, dibutyryl cyclic AMP (db-cAMP) and fatty acid-free bovine serum albumin were from Sigma (Deisenhofen, Germany). Fura-2 pentakis(acetoxymethyl) ester (Fura-2/AM) was from Molecular Probes (Eugene, Ore., USA), 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA/AM) from Calbiochem (Bad Soden, Germany) and pertussis toxin (PTX) from List Biological Laboratories (Campbell, Calif., USA). Myo-[3H]inositol (24.4 Ci/mmol) and [32P]NAD (800 Ci/mmol) were from Biotrend (Köln, Germany). Polyvinylpyrrolidone-free polycarbonate filter membranes were from Nucleopore (Tübingen, Germany), and staining solution Diff-Quick was from Baxter (Düdingen, Switzerland). Purified Clostridium difficile toxin B was kindly donated by Dr. C. von Eichel-Streiber (Universität Mainz, Germany). Cell culture and toxin treatments. J82 cells obtained from the American Type Culture Collection were cultured in MEM growth

medium containing 10% fetal calf serum, 100 units/ml penicillin G and 100 µg/ml streptomycin. The cells were grown to near confluence in 145 mm culture dishes in a humidified atmosphere of 5% CO2 at 37° C. Before experiments, cells were treated for 24 h with and without PTX or toxin B at the indicated concentrations. Measurement of [Ca2+]i. Cytosolic free Ca2+ concentration ([Ca2+]i) was determined with the fluorescent calcium indicator dye Fura-2 (Grynkiewicz et al. 1985) in a Perkin-Elmer spectrofluorometer with a water jacket maintaining the temperature at 37° C. Confluent J82 cells were detached from the culture dishes by exposure to Puck’s salt solution A/EDTA (135 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 5 mM D-glucose, 1.5 mM EDTA) and loaded with 2 µM Fura-2/AM at 37° C in culture medium containing 10 mg/ml bovine serum albumin. After 1 h incubation, cells were pelleted and resuspended at a density of 1.2–3.0 × 105 cells/ml in fresh serumfree culture medium. Cells were transferred to Hank’s balanced salt solution (HBSS), containing 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 5 mM D-glucose, buffered at pH 7.4 with 20 mM HEPES, directly before use for fluorescence measurements as described before (Schmidt et al. 1995). Data shown represent maximal increases (peak) in [Ca2+]i after stimulation with receptor agonist. Measurement of inositol phosphate accumulation. Phospholipase C (PLC) activity in intact J82 cells was determined essentially as described before (Schmidt et al. 1995). In brief, J82 cells were plated at a density of 3000 cells/cm2 in 35 mm culture dishes and grown to subconfluence for 72 h in culture medium containing 10% fetal calf serum. Cellular phosphoinositides were labeled by incubating monolayers of cells for 48 h with myo-[3H]inositol (1 µCi/ml) in inositol-free medium 199 supplemented with 3.5 mg/ ml bovine serum albumin, 2 mM L-glutamine, 5 µg/ml transferrin and 0.12 units/ml insulin. Thereafter, the labeling medium was replaced, and the cells were equilibrated for 10 min at 37° C in HBSS and then incubated for another 10 min at 37° C with 10 mM LiCl in HBSS. Finally, cells were stimulated with the receptor agonists in a final volume of 1 ml for 10 min at 37° C. Total [3H]inositol phosphates formed were separated by anion exchange (AG 1-X8, BioRad) column chromatography as described (Berridge et al. 1982). Radioactivity in the eluates was determined by liquid scintillation counting. Protein was measured by the method of Bradford (1976), using γ-globulin as a standard. [3H]Inositol phosphate formation is normalized for protein content and is given as percent of basal [3H]inositol phosphate accumulation. Measurement of cell motility. The bottom wells of a 48 well microchemotaxis chamber (NeuroProbe, Cabin John, M., USA) were filled with 25 µl serum-free MEM containing the receptor agonists and were carefully covered with a polyvinylpyrrolidone-free polycarbonate filter membrane with a pore size of 8 µm. After adjustment of the top plate, 50 µl of a cell suspension in serum-free MEM at a density of 1 × 106 cells/ml were applied to the upper wells. The cell suspension was prepared from confluent cell monolayers grown in 145 mm culture dishes. After 4 h incubation in a humidified atmosphere of 5% CO2 at 37° C, the filter membrane was removed from the chemotaxis chamber, and non-migrated cells were wiped off by drawing the filter membrane carefully over a wiper blade. The filter membrane was air-dried and stained in Diff-Quick staining solution according to the manufacturer’s instructions. Migrated cells were quantified on the lower side of the filter by counting three random areas of each well under a microscope with a magnification of 320-fold (high power field). PTX-catalyzed ADP-ribosylation. After 24 h treatment with and without PTX, J82 cells (1 × 106) were incubated for 1 h at 4° C in 50 µl of a buffer, containing 0.5% (w/v) Lubrol, 1 mM EDTA, 0.6 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 µM leupeptin, 200 µM soybean trypsin inhibitor, 3 mM benzamidine and 10 mM Tris-HCl, pH 7.5. Insoluble components were pelleted by centrifugation for 15 min at 13 000 × g, and the supernatant (20 µl) was analyzed for its content of ADP-ribosylated G pro-

771 teins. The ADP-ribosylation reaction was carried out as described before (Wieland et al. 1996) in a reaction mixture (50 µl) containing 50 mM triethanolamine-HCl, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, 1 mM ATP, 100 µM GDP and 100 nM [32P]NAD (1 µCi/tube). The reaction started by addition of 0.2 µg/tube preactivated PTX was for 30 min at 30° C and was stopped by addition of 60 µl sample buffer (Laemmli 1970). Samples were heated for 5 min at 95° C and analyzed by SDS-PAGE (12.5% polyacrylamide) and autoradiography, which was quantified by densitometric analysis, using the QuantiScan program (Biosoft). Data presentation and analysis. Data shown are means ± SD from a representative experiment performed in triplicate, repeated at least twice with similar results. Concentration response curves were analyzed using iterative non-linear regression analysis (InPlot program, GraphPAD Software, San Diego, Calif., USA).

Results Expression of G protein-coupled, calcium-mobilizing receptors in J82 cells To identify possible candidate G protein-coupled receptors stimulating migration of human transitional-cell carcinoma cells, we first studied the expression of calciummobilizing receptors in J82 cells. Out of many different agonists studied, significant increases in [Ca2+]i, measured with the fluorescent Ca2+ indicator dye Fura-2, were observed upon stimulation of J82 cells with LPA, thrombin, bradykinin, bombesin and histamine. [Ca2+]i increases induced by all of these five agonists were characterized by a rapid and transient increase, followed by a lower plateau phase (data not shown). As illustrated in Fig. 1 for maximally effective concentrations of these agonists, the highest [Ca2+]i increase, by more than 300 nM (330 ± 53 nM), was observed following application of bombesin (1 µM), followed by LPA (1 µM), bradykinin (1 µM) and histamine (1 mM), all of which increased [Ca2+]i by 200–250 nM, while thrombin (1 U/ml) was least effective, inducing

Fig. 2 Agonist-induced inositol phosphate formation in J82 cells. [3H]Inositol phosphate formation was determined in myo-[3H]inositol-labeled J82 cells in response to LPA (1 µM), thrombin (1 U/ml), bradykinin (1 µM), bombesin (1 µM) and histamine (1 mM) as described in “Materials and methods”. [3H]Inositol phosphate formation is given as % of basal values, amounting to 12.9 ± 2.7 × 103 cpm/mg protein

a maximal [Ca2+]i increase of only ~ 50 nM. To study the G protein subtype involved in the action of these agonists, [Ca2+]i responses were monitored in J82 cells pretreated for 24 h with 100 ng/ml pertussis toxin (PTX). As shown below, this treatment was supramaximally effective in ADP-ribosylating endogenous Gi type G proteins in J82 cells. PTX pretreatment did not affect the [Ca2+]i responses to either bombesin, bradykinin, histamine or thrombin, whereas the stimulatory effect of LPA was reduced by ~ 50% ([Ca2+]i increases of 224 ± 8 nM and 103 ± 6 nM in control and PTX-treated cells, respectively). To determine whether the calcium-mobilizing agonists stimulate phosphoinositide-hydrolyzing PLC, formation of inositol phosphates in response to the agonists was measured. All agonists studied caused an increase in inositol phosphate accumulation in J82 cells (Fig. 2). There was, however, no quantitative correlation between maximal [Ca2+]i responses and inositol phosphate formation. Histamine (1 mM) was most effective, inducing an about 10-fold increase in inositol phosphate accumulation, followed by bradykinin (1 µM, 6- to 7-fold increase), thrombin (1 U/ml, about 4-fold increase), LPA (1 µM) and bombesin (1 µM), the latter two increasing inositol phosphate accumulation by only about 2-fold.

Receptor regulation of J82 cell motility

Fig. 1 Agonist-induced increases in [Ca2+]i in J82 cells; influence of PTX. J82 cells were pretreated for 24 h without (open bars) and with (filled bars) 100 ng/ml PTX. Thereafter, peak increases in [Ca2+]i were determined in Fura-2-loaded cells in response to LPA (1 µM), thrombin (1 U/ml), bradykinin (1 µM), bombesin (1 µM) and histamine (1 mM) as described in “Materials and methods”. Basal [Ca2+]i was 137 ± 11 nM and 96 ± 21 nM (n = 6) in control and PTX-treated cells, respectively

To examine whether the identified calcium-mobilizing receptors promote cell motility, we measured migration of J82 cells in a micro-chemotaxis chamber. Strong motile responses were observed upon stimulation of J82 cells with LPA and thrombin (Fig. 3). Migration induced by LPA was usually less (40–80%) than that caused by thrombin. The number of cells migrating through the filter membranes in response to the two agonists was 5- to 20fold that of control basal migration. In contrast to LPA and thrombin, the other three calcium-mobilizing receptor

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being obtained at about 0.5 µM (EC50 0.67 ± 0.16 µM) and 20 µM LPA, respectively (Fig. 4B). Mechanisms involved in receptor-stimulated migration of J82 cells

Fig. 3 Agonist-induced motility of J82 cells. Migration of J82 cells was determined in a micro-chemotaxis chamber without (Basal) and with LPA (20 µM), thrombin (10 U/ml), bradykinin (100 µM), bombesin (1 µM) and histamine (100 µM) as indicated

First, the type of G proteins mediating the motility responses to thrombin and LPA receptor activation was examined. In cells pretreated with PTX (100 ng/ml, 24 h), stimulation of motility by thrombin was not altered compared to control cells. Both the maximal response and the EC50 value (1.9 ± 0.01 U/ml) were virtually identical to those of untreated cells (Fig. 4A). In contrast, PTX abolished the LPA-induced migration of J82 cells (Fig. 4B). Pretreatment of J82 cells for 24 h with as low as 0.1 ng/ml PTX completely inhibited cell motility induced by LPA (20 µM), and half-maximal inhibition was observed at about 10 pg/ml PTX (Fig. 5A). This high sensitivity to PTX raised the question whether PTX may cause the inhibition independent of its action on Gi type G proteins.

Fig. 4A, B Concentration-response curves of thrombin- and LPAstimulated motility of J82 cells; influence of PTX. J82 cells were pretreated for 24 h without (p) and with (P) 100 ng/ml PTX. Thereafter, cell migration in response to (A) thrombin and (B) LPA at the indicated concentrations was determined

agonists, i.e., bradykinin, bombesin and histamine, failed to stimulate motility of J82 cells at any concentration studied. Stimulation of J82 cell motility by thrombin and LPA was concentration-dependent. Thrombin increased motility with an EC50 value of 2.1 ± 0.06 U/ml, and maximal stimulation was observed at 10 U/ml thrombin (Fig. 4A). Motility stimulation by LPA was observed at concentrations ≥ 10 nM, with half-maximal and maximal increases

Fig. 5A, B Concentration-dependent effect of PTX on LPA-induced motility and ADP-ribosylation of Gi proteins in J82 cells. J82 cells were pretreated for 24 h with the indicated concentrations of PTX. Thereafter, (A) cell migration was determined in the absence (p) and presence of 20 µM LPA (P), and (B) PTX-catalyzed [32P]ADP-ribosylation of Gi proteins was measured in detergent extracts. The inset in B shows a representative autoradiogram, with the molecular weight markers on the left and the PTX concentrations (ng/ml) used for treatment of intact J82 cells at the bottom

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However, this was apparently not the case. First, acute addition of PTX (1, 3 and 10 ng/ml) to J82 cells only 30 min before LPA did not alter the LPA response (data not shown). Second, PTX caused incorporation of [32P]ADPribose into one protein band of ~ 40 kDa in detergent extracts of control cells (Fig. 5B, inset). In extracts of cells pretreated with PTX, this PTX-catalyzed [32P]ADP-ribosylation of the 40 kDa protein(s) was strongly reduced. In agreement with the functional data, pretreatment of J82 cells for 24 h with as low as 0.01 and 0.1 ng/ml PTX reduced (by ~ 70%) and almost completely abolished, respectively, the back-[32P]ADP-ribosylation (Fig. 5B), indicating that the Gαi proteins are covalently modified by prior PTX treatment of intact cells. As signal transduction of different receptors inducing cell motility has been reported to converge on one or more members of the Rho family of small molecular weight G proteins (Hall 1994; Takai et al. 1995; Zigmond 1996), we studied the effect of Clostridium difficile toxin B, which has recently been reported to glucosylate and thereby inactivate Rho, Rac and Cdc42 G proteins (Just et al. 1994, 1995), on J82 cell migration. As observed in various other cell types (Just et al. 1994, 1995; Schmidt et al. 1996), treatment of J82 cells with toxin B induced rounding-up of the cells (data not shown), indicating disruption of the actin cytoskeleton. The motility response of J82 cells to LPA (10 µM) was completely blocked by prior treatment of the cells with toxin B (100 pg/ml, 24 h) (Fig. 6, left panel). The same toxin B treatment decreased the thrombin (10 U/ml)-induced motility by 70%. Similar data were observed in cells pretreated with cytochalasin B (5 µg/ml, 30 min), an agent causing direct disruption of the actin cytoskeleton (Fig. 6, right panel). Since LPA and thrombin stimulated PLC and increased [Ca2+]i in J82 cells, we examined the effects of PMA, a direct activator of protein kinase C (PKC), and BAPTA/ AM, a membrane-permeable Ca2+-chelating agent, on cell motility. Pretreatment of J82 cells with BAPTA/AM (20 µM, 30 min) completely suppressed the [Ca2+]i responses to LPA and thrombin (Fig. 7A), without affecting basal [Ca2+]i (data not shown). The same treatment also strongly attenuated the motility response of J82 cells to the receptor agonists (Fig. 7B). Motility induced by LPA (20 µM)

Fig. 6 Influence of toxin B and cytochalasin B on agonist-induced motility of J82 cells. J82 cells were pretreated for 24 h without (open bars) and with (filled bars) 100 pg/ml Clostridium difficile toxin B (left panel) or for 30 min without (0.1% dimethylsulfoxide as solvent, open bars) and with (filled bars) 5 µg/ml cytochalasin B (right panel). Thereafter, cell migration was determined without (Basal) and with 10 µM LPA or 10 U/ml thrombin

Fig. 7A, B Influence of BAPTA/AM on [Ca2+]i responses and agonist-stimulated motility of J82 cells. J82 cells were pretreated for 30 min with (filled columns) and without (open columns) 20 µM BAPTA/AM. Thereafter, (A) peak [Ca2+]i increases in response to 20 µM LPA (left panel) and 10 U/ml thrombin (right panel) were determined, and (B) cell motility was determined in the absence (Basal) and presence of 20 µM LPA or 10 U/ml thrombin

Fig. 8A, B Influence of PMA on agonist-stimulated motility and [Ca2+]i responses of J82 cells. J82 cells were pretreated for 30 min with (filled columns) and without (open columns) 100 nM PMA. Thereafter, (A) cell motility was determined in the absence (Basal) and presence of 20 µM LPA or 10 U/ml thrombin and (B) peak [Ca2+]i increases in response to 20 µM LPA (left panel) and 10 U/ ml thrombin (right panel)

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Fig. 9 Influence of forskolin and db-cAMP on agonist-stimulated motility of J82 cells. J82 cells were pretreated for 30 min without (open columns) and with 30 µM forskolin (filled columns) or 1 mM db-cAMP (hatched columns). Thereafter, cell motility was determined in the absence (Basal) and presence of 20 µM LPA or 10 U/ml thrombin

was virtually abolished, and that induced by thrombin (10 U/ml) was reduced by about 60%. The phorbol ester PMA by itself did not induce migration of J82 cells. In contrast, when cells were pretreated with PMA (100 nM, 30 min), the stimulatory effects of LPA (20 µM) and thrombin (10 U/ml) were reduced by 70–90% (Fig. 8A). Half-maximally inhibitory effects on the LPA- and thrombin-stimulated motility were observed at about 10 nM PMA (data not shown). In order to get some hint on the process inhibited by PMA, the effect of PMA on [Ca2+]i increases induced by LPA (20 µM) and thrombin (10 U/ml) was studied. Pretreatment of J82 cells with PMA (100 nM, 30 min) almost completely abolished the [Ca2+]i increases induced by either LPA or thrombin (Fig. 8B). LPA-stimulated invasion of rat ascites hepatoma cells has been reported to be inhibited by cyclic AMP (Imamura et al. 1993). To study a possible regulation of J82 cell migration by cyclic AMP, agonist-stimulated motility was examined in cells pretreated for 30 min with the direct adenylyl cyclase activator, forskolin (30 µM), and the membrane-permeable cyclic AMP analogue, db-cAMP (1 mM). As illustrated in Fig. 9, these treatments did not affect thrombin (10 U/ml)-stimulated motility. However, both forskolin and db-cAMP inhibited, by 50–60%, stimulation of J82 cell migration by LPA (20 µM). Neither forskolin nor db-cAMP reduced the [Ca2+]i increases induced by either LPA or thrombin (data not shown).

Discussion Aim of the present study was to identify receptors for soluble extracellular molecules stimulating motility of human transitional-cell carcinoma cells. Therefore, first the expression of receptors, specifically of calcium-mobilizing receptors, was examined in J82 cells, as various migration-inducing agents in other cell types have been shown to cause an increase in intracellular calcium concentration (Stoker and Gherardi 1991; Stossel 1993). Out

of many different agonists studied, rapid and transient increases in [Ca2+]i in J82 cells were observed upon stimulation with LPA, thrombin, bradykinin, bombesin and histamine. There were, however, large differences between these five agonists in maximal [Ca2+]i increases. Most notably, the maximal increase in [Ca2+]i induced by thrombin (about 50 nM) was by far much smaller than that induced by the other agonists, causing [Ca2+]i increases of 200–350 nM. All of the five calcium-mobilizing agonists studied also induced PLC stimulation. There was, however, no quantitative correlation between the two responses, i.e., the maximal increases in [Ca2+]i and inositol phosphate formation. For example, bombesin most efficiently increased [Ca2+]i (by ~ 350 nM), whereas its stimulatory effect on inositol phosphate formation was rather small, only about 2-fold, while on the other hand thrombin increased [Ca2+]i by only ~ 50 nM but caused an about 4fold increase in inositol phosphate formation. The reason for this discrepancy was beyond the aim of the present study, however, the data strongly suggest that the extent of inositol phosphate accumulation is not the sole determinant of receptor regulation of [Ca2+]i in J82 cells. Of the five agonists studied, known to act via G protein-coupled receptors, only the stimulatory effect of LPA on [Ca2+]i and inositol phosphate formation (data not shown) was partially (~ 50%) sensitive to PTX treatment. These data indicate that the putative LPA receptor couples in J82 cells to both Gi and Gq type G proteins, whereas the [Ca2+]i and PLC responses induced by thrombin, bradykinin, bombesin and histamine receptor activation is solely mediated by PTX-insensitive, most likely Gq type G proteins. Coupling of the LPA receptor to Gi and Gq type G proteins is well known from studies in fibroblasts and other cell types (Moolenaar 1995). Interestingly, of the five calcium-mobilizing, bona fide G protein-coupled receptors identified in J82 cells, only two exhibited migration stimulatory activity. Bradykinin, bombesin, and histamine, which have been reported to stimulate migration of other tumor cells (Ruff et al. 1985; Tilly et al. 1990), were ineffective. In contrast, activation of LPA and thrombin receptors potently stimulated motility of the human transitional-cell carcinoma cells. This distinct effectiveness of LPA and thrombin was not due to the fact that these two agonists were more effective than the other agents in inducing [Ca2+]i increase or PLC stimulation. For example, LPA was a rather weak PLC stimulant, and, as mentioned above, thrombin was least effective in calcium mobilization. Furthermore, there was also no major difference between the five agonists in inducing Ca2+ release from internal stores and causing Ca2+ influx from the extracellular milieu. Chelation of extracellular Ca2+ by EGTA reduced the peak [Ca2+]i increases by about 50% and abolished the plateau phase of [Ca2+]i increase by any of the five agonists studied (data not shown). These data clearly indicate that PLC stimulation, including rise in [Ca2+]i, is not sufficient for initiating receptor stimulation of J82 cell motility. The G protein subtype mediating the LPA and thrombin receptor stimulation of J82 cell motility was studied

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by examining the sensitivity to PTX, which uncouples Gi type G proteins from receptors. Whereas the thrombinstimulated motility was not affected by PTX treatment, the stimulatory effect of LPA was completely abrogated in PTX-treated cells. J82 cells exhibited a very high sensitivity to PTX, with half-maximal and maximal inhibition being observed after 24 h treatment with as low as 10 pg/ml and 0.1–1 ng/ml PTX, respectively. This high functional sensitivity correlated with a similar high sensitivity of ADP-ribosylation of endogenous Gαi proteins by PTX. These data, thus, indicate that coupling of LPA and thrombin receptors to the migration-inducing machinery in the human transitional-cell carcinoma cells is mediated by two distinct G proteins, i.e., PTX-sensitive Gi type and PTX-insensitive Gq or G12 type G proteins, respectively. In contrast to J82 cells, invasion of rat ascites hepatoma cells induced by LPA was reported to be PTX-insensitive (Imamura et al. 1993). On the other hand, Offermanns et al. (1997) recently reported that migration of fibroblastlike cells from E8.5 embryos in response to thrombin, which was by about 50% PTX-sensitive, is abrogated in cells deficient in Gα13, a member of the G12 family. Thus, G protein coupling of LPA and thrombin receptors to the migratory response appears to be different in different cell types. Receptor stimulation of cell motility apparently involves a wide variety of distinct proteins, most notably actin and actin-binding and -regulatory proteins, as well as various G proteins, specifically members of the Rho family of small molecular weight G proteins (Hall 1994; Takai et al. 1995; Huttenlocher et al. 1995; Lauffenburger and Horwitz 1996; Zigmond 1996). As demonstrated herein, these proteins also play a pivotal role in agonist-stimulated motility of J82 cells. First, direct inhibition of actin assembly by cytochalasin B largely prevented the stimulatory action of LPA and thrombin. Second, in agreement with findings reported with the Rho-inactivating Clostridium botulinum C3 exoenzyme on motility of other cell types (Stasia et al. 1991; Imamura et al. 1993; Takaishi et al. 1993, 1994), agonist-stimulated motility of J82 cells was strongly inhibited upon inactivation of Rho family G proteins by Clostridium difficile toxin B. These findings indicate that Rho G proteins play a central role in the motility stimulatory actions of LPA and thrombin in J82 cells as well. Thus, it may be speculated that the failure of the other calcium-mobilizing receptors (bradykinin, bombesin, histamine) identified in J82 cells to induce cell movement may be due to the ineffectiveness of these receptors to activate Rho proteins in J82 cells, a hypothesis presently under investigation in our laboratory. In addition to many protein factors involved in cell migration, calcium plays a critical role in cell motility (Korn 1978; Stossel, 1993; Huttenlocher et al. 1995). Calcium is known to directly activate some of the actin escort proteins and is required for myosin phosphorylation, regulating myosin’s assembly and contractile activity with actin filaments. Therefore, it was not surprising to observe that preventing the calcium transients induced by LPA and thrombin by intracellular calcium chelation with BAPTA/

AM blunted the agonist-induced motility. Thus, although thrombin induced only a very modest increase in [Ca2+]i, this increase seemed to be required for stimulated motility. Alternatively, the BAPTA/AM treatment may have prevented [Ca2+]i fluctuations apparently occurring during and required for cell migration, as recently reported for migration of mice cerebellar granule cells (Komuro and Rakic 1996). As LPA and thrombin stimulated PLC in J82 cells, we addressed the question whether activation of PKC, a target of PLC-formed diacylglycerol, is involved in agonist-stimulated cell motility. Direct PKC activation by the phorbol ester PMA did not induce J82 cell motility. In contrast, short term pretreatment of J82 cells with PMA blunted the mobility response of both receptor agonists, suggesting participation of a PKC-sensitive target(s) in receptor-stimulated cell motility. As PMA has been reported to inhibit receptor-mediated PLC stimulation and calcium mobilization in several cell types (Della Bianca et al. 1986; Brown et al. 1987; Hepler et al. 1988; VázquezPrado and García-Sáinz 1996; Kawabata et al. 1996), we examined whether PMA may inhibit cell migration by inhibiting the calcium response. Indeed, PMA treatment abolished the [Ca2+]i increases induced by either LPA or thrombin, indicating that at least one target involved in PMA-induced inhibition of J82 cell migration is the rise in [Ca2+]i. Another potential target for PMA could be nchimaerin, which was recently identified as a phorbol ester and LPA target and which acts as a Rac GTPase-activating protein (Ahmed et al. 1993). Finally, to potentially identify further distinct signalling pathways for LPA and thrombin receptor-stimulated motility, we examined the effect of cAMP elevation on J82 cell motility. There was a clear distinction of the two agonist responses. In contrast to the motility response induced by thrombin which was unaffected, stimulation of J82 cell motility by LPA was strongly inhibited (50–60%) by prior cell treatment with either forskolin or db-cAMP. Similar data have been reported for LPA-stimulated invasion of rat ascites hepatoma cells, which response, in contrast to the LPA-stimulated motility of J82 cells, was PTX-insensitive (Imamura et al. 1993). In conclusion, we demonstrate in the present study that human transitional-cell carcinoma (J82) cells express receptors for LPA and thrombin, which potently and efficiently stimulate migration of these cells. Both agonists, known to act on G protein-coupled receptors, also increased [Ca2+]i and stimulated PLC. However, these primary responses by themselves apparently do not suffice for initiating cell motility, as activation of other receptors identified in J82 cells, i.e., those for bradykinin, bombesin and histamine, also increased [Ca2+]i and PLC activity, partially even more efficiently than LPA or thrombin, but failed to stimulate J82 cell motility. The complex signalling pathways leading to stimulated motility upon activation of LPA and thrombin receptors appear to involve some common, apparently essential factors, such as [Ca2+]i changes, Rho family small molecular weight G proteins and reorganization of the actin cytoskeleton, as well as some distinct components, most notably distinct subtypes

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of heterotrimeric G proteins and apparently also distinct cAMP-sensitive targets. Further investigations into the signalling pathways leading to stimulated migration of human transitional-cell carcinoma cells by the serum constituents, LPA and thrombin, may finally provide potential targets for therapy of bladder carcinomas. Acknowledgements We would like to thank Dr. C. von EichelStreiber for the kind gift of Clostridium difficile toxin B. This work was supported by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft to G.L. and the IFORES program of the Universitätsklinikum Essen.

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